Activation of natural cytotoxicity (killing of target cells) by NK cells requires combinations of receptor pairs that deliver synergistic signals for activation of intracellular Ca2+ release, cytokine production, and cytotoxicity. The main question we have addressed is how distinct signals from co-activation receptors are integrated to achieve synergy, and which molecular checkpoint(s) control this process. Among the receptor combinations that provide synergistic activation in resting NK cells are NKG2D (CD314) and 2B4 (CD244). Upon stimulation with its natural ligands, such as ULBP molecules, NKG2D recruits phosphatidylinositol-3-kinase (PI3K) and Grb2-Vav1 through its association with DAP10. 2B4 binds to its ligand CD48, which is expressed on hematopoietic cells, and recruits the small adaptor SAP through cytoplasmic tyrosine-based motifs. SAP binds to the tyrosine kinase Fyn. Because synergy requires the integration of such diverse signals, understanding the basis for synergy would disclose how disparate signals converge to a certain point at which synergy occurs.&#8232;Synergistic NK cell activation by two coactivation receptors was accompanied by enhanced Vav1 phosphorylation, which was equivalent to the sum of phosphorylation induced by each receptor alone and was required to overcome inhibition by c-Cbl ubiquitin ligase. Until recently, there was no information about how distinct signals from synergizing receptors converge to regulate Vav1 and its downstream signaling. Based on the additive, rather than synergistic phosphorylation of Vav1 during synergy, we hypothesized that different pools of Vav1 may complement each other to achieve synergy. As a first step, we tested the role of adaptors known to contribute to signaling for lymphocyte activation. We investigated whether adaptor proteins such as SLP-76 and linker for activation of T cells (LAT) are required for NK cell synergy. SLP-76 and LAT are important adaptors that form the backbone of signaling complexes T cells. &#8232;Synergy among receptors is best achieved by complementation of independent signals. Stimulation of natural killer (NK)-cell cytotoxicity and cytokine secretion requires synergistic signals from coactivation receptors. Our previous work has shown that synergy is required to overcome inhibition of guanine exchange factor Vav1-dependent signals by the ubiquitin ligase c-Cbl. Whether complementation of unique signals is involved and, if so, at what level, is unknown. We have shown here that the adapter SLP-76 is required for the synergy and that each receptor of a synergistic pair controls the phosphorylation of a specific tyrosine in SLP-76. Each one of the two phosphorylated tyrosines that constitute separate binding sites for Vav1 is required for synergistic mobilization of Ca2+. The selective phosphorylation of either tyrosine 113 or tyrosine 128 in SLP-76 is unique to signaling by natural cytotoxicity co-activation receptors, as stimulation of NK cells by the Fc receptor CD16 resulted in phosphorylation at both sites, much like T cells stimulated by T-cell receptor. Our data reveal an unexpected degree of selectivity in the phosphorylation of two SLP-76 tyrosines by NK cell co-activation receptors, suggest that binding of two Vav1 molecules to SLP-76 may have a synergistic effect on Vav1 function, and show that triggering of NK cell effector function is tightly regulated by complementary signals.&#8232; Mendelian analysis of disorders of immune regulation can provide molecular insight into pathways utilized for host defense and immune tolerance. Three families with a dominantly inherited complex of cold urticaria, antibody deficiency, and susceptibility to infection and autoimmunity were identified by the group of Josh Milner. The phenotype is due to mutations in PLCG2, encoding a signaling molecule expressed in B, NK, and mast cells. Using immunophenotyping by flow cytometry, we showed that NK cell degranulation, and ligand-induced calcium flux was impaired in B cells and NK cells, but in T cells. The deletions in PLCG2, located within an autoinhibitory domain, produce protein products with constitutive phospholipase activity. Genomic deletions in PLCG2 cause gain of phospholipase C-gamma2 function leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and impaired immune function.